慢病毒介导的siRNA靶向干扰EIF4G1鼻咽癌稳定细胞株的建立

目的 检测人真核翻译起始因子4G1(EIF4G1)基因在8株鼻咽癌细胞中的差异表达,寻找最高表达的细胞株.构建EIF4G1基因的小干扰RNA(siRNA)表达载体,建立稳定干扰EIF4G1表达的鼻咽癌细胞株,测定干扰效率.方法 应用荧光定量PCR检测EIF4G1 mRNA在鼻咽癌细胞株5-8F、6-10B、C666-1、CNE1、CNE2、HNE1、HONE1、SUNE1表达水平.构建重组靶向EIF4G1 shRNA慢病毒表达质粒pLenti6/BLOCK-iT-DEST/EIF4G1shRNA.用293FT细胞包装后产生的成熟慢病毒颗粒感染5-8F细胞.经杀稻瘟菌素筛选后,建立稳定表达siRNA的5-8F鼻咽癌细胞株.最后荧光定量PCR检测干扰效率.结果 在8个鼻咽癌细胞株中,EIF4G1显示在5-8F细胞株中表达最高.PCR和测序验证pLenti6/BLOCK-iT-DEST/EIF4G1shRNA重组质粒构建成功;经293FT细胞病毒包装,感染5-8F细胞后,与阴性对照组和未十扰组相比可明显抑制EIF4G1 mRNA水平EIF4G1表达.结论 成功构建TpLenti6/BLOCK-iT-DEST/EIF4G1shRNA慢病毒重组质粒,建立了稳定靶向干扰EIF4G1表达的siRNA5-8F鼻咽癌细胞株.

Establishment of a stable nasopharyngeal carcinoma cell line with lentivirus-mediated RNA interference for EIF4G1 gene silencing

Objective To establish a nasopharyngeal carcinoma (NPC) cell line with stable EIF4G1 gene silencing induced by small interfering RNA (siRNA). Methods The EIF4G1 mRNA levels in 8 NPC cell lines including 5-8F,6-10B,C666-1,CNE1,CNE2,HNE1,HONE1,and SUNE1 were detected by fluorescence quantitative RT-PCR (QRT-PCR). The recombinant lentivirus shRNA expression plasmid targeting EIF4G1 gene was packaged into mature lentivirus by 293FT cells and used to infect 5-8F cells. After blasticidin selection of NPC cells wi...